Diabetes is a syndrome of disordered metabolism, usually due to a combination of hereditary and environmental causes, resulting in abnormally high blood sugar levels (hyperglycemia). Blood glucose levels are controlled by a complex interaction of multiple chemicals and hormones in the body, including the hormone insulin made in the beta cells of the pancreas. Diabetes mellitus refers to the group of diseases that leads to high blood glucose levels due to defects in either insulin secretion or insulin action in the body.
Diabetes develops due to a diminished production of insulin (in type 1) or resistance to its effects (in type 2 and gestational). Both lead to hyperglycemia, which largely causes the acute signs of diabetes, namely excessive urine production, resulting compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism.
The injections by a syringe, insulin pump, or insulin pen deliver insulin, which is a basic treatment of type 1 diabetes. Type 2 diabetes is managed with a combination of dietary treatment, exercise, medications and insulin supplementation. All forms of diabetes have become treatable since insulin became medically available, but there is still no cure.
According to a World Health Organization report in 2000, at least 171 million people worldwide were suffering from diabetes, or 2.8% of the population. Its incidence is increasing rapidly, however, and it is estimated that by the year 2030, this number will almost double. Diabetes mellitus occurs throughout the world, but is more common (especially type 2) in the more developed countries. The greatest increase in prevalence is, however, expected to occur in Asia and Africa, where most patients will likely be found by 2030.
Glucagon-like peptide (GLP-1), gastric inhibitory peptide (GIP), glucagon and ghrelin have been reported as peptide biomarkers of metabolic diseases such as diabetes. The major source of GLP-1 in the body is found in the intestines. The typical normal blood concentration of GLP-1 in circulation is in a range of 3-85 picomolar. GLP-1 possesses several physiological properties that make it a subject of intensive investigation as a potential treatment of diabetes. Gautier, et al., Diabetes Met. 31:233-42 (2005). GLP-1 is known to increase insulin secretion from the pancreas, decrease glucagon secretion from the pancreas, increase beta cell mass and insulin gene expression, inhibit acid secretion and gastric emptying in the stomach, and decrease food intake by increasing satiety. Baggio, et al., J. Gastroenterol. 132:2131-57 (2007). Once in circulation, however, GLP-1 has been reported to exhibit a short biological half-life of about 1.5-5 minutes (Hui, et al., Eur. J. Endocrinol. 146:863-9 (2002)), due to proteolytic degradation caused by the proteases including dipeptidyl peptidase (DPP)-IV.
The active form of GIP is a 42-amino acid polypeptide represented by the sequence: YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDKHNITGQ (“GIP(1-42)”). GIP(1-42) is synthesized by K cells which are found in the mucosa of the duodenum and the jejunum of the gastrointestinal tract. It is believed that GIP(1-42) induces insulin secretion via a mechanism that involves interaction between GIP and 7 transmembrane GIP(1-42) receptors on pancreatic beta-cells. The normal fasting concentration of GIP(1-42) in plasma is about 6-12 pmol/L, whereas the normal non-fasting concentration is about 80-300 pmol/L. GIP(1-42) has been reported to exhibit a half-life in circulation of about 5 minutes.
Glucagon, a 29-amino acid peptide, is involved in carbohydrate metabolism. Produced by the pancreas, it is released when the glucose level in the blood is low (hypoglycemia). It binds to receptors on liver cells (hepatocytes), causing the liver to convert stored glycogen into glucose and release then release the glucose into the bloodstream. As these stores become depleted, glucagon then stimulates synthesis of additional glucose in the liver. The action of glucagon is thus opposite that of insulin, which instructs the cells in the body to take in glucose from the blood. Glucagon also regulates the rate of glucose production through a process known as lipolysis. Typical normal blood concentration of glucagon in circulation is 11-17 picomolar. Once in circulation, glucagon has a half-life of about 8-18 minutes.
Ghrelin is a hormone produced mainly by P/D1 cells lining the fundus of the human stomach and epsilon cells of the pancreas that stimulates appetite. Typical normal blood concentration is also in the picomolar range. Ghrelin is a 28-amino acid peptide having the sequence NH2-GSSFLSPEHQRVQQRKESKKPPAKLQPR-COOH. One of the biologically active forms of this peptide, known as acylated ghrelin, contains an n-octanoyl group on Ser3 (i.e., —CH3(CH2)6COO—). This peptide exerts endocrine actions such as stimulation of Growth Hormone (GH) release from the pituitary gland, and various physiological effects such as induction of adiposity (increase in fat tissue) and body weight gain due to appetite-stimulating effects and increased food intake, and stimulation of gastric acid secretion and motility. Kojima, et al., Trends Endocrinol. Metab. 12:118-122 (2001); Kojima, et al., Physiol. Rev. 85:495-532 (2005). Thus, in addition to diabetes, acylated ghrelin is a known metabolic biomarker for related conditions such as diet-induced weight loss and fasting. Another form of ghrelin peptide is known as des-acyl-ghrelin, which is a metabolically inactive form having its own functions including the modulation of cell proliferation (Baldanzi, et al., J. Cell Biol. 159:1029-37 (2002); Ariyasu, et al., Endocrinol. 2005:355-64 (2005)) and adipogenesis (Muccioli, et al., Eur. J. Pharmacol. 498:27-35 (2004)). Normal plasma concentration of ghrelin, including both active and inactive forms, ranges from about 300 to about 700 pg/ml (or about 0.08 to about 0.19 nM or about 0.09 to about 0.19 fmol/μl), and fluctuates with time. The major circulating form of ghrelin is dec-acyl ghrelin [Hosoda, et al., Biochim. Biophys. Res. Comm. 279:909-13 (2000)], and thus the majority of this amount is not in the form of the more accurate metabolic biomarker. Once in circulation, ghrelin has a half-life of about 30 minutes.
Yi, et al., J. Proteome Res. 6(5):1768-81 (2007), for example, reports that proteolytic degradation of serum and plasma proteins caused by intrinsic proteases occurs during the first minutes of sample collection and handling (which suggests rapid ex vivo proteolytic degradation). In a subsequent publication, Yi et al., J. Proteome Res. 7(12):5112-8 (2008), reports that although the discovery of disease markers in blood fluid continues to accelerate as proteomics technology becomes both more powerful and more widely available, there has been notably less success in transitioning these discoveries into clinical utility, spurring a growing interest in understanding the barriers to this transition. Aside from the short half-lives and the rather small concentrations of GLP-1, GIP, glucagon and ghrelin (and particularly the biologically active forms thereof) in plasma, complications arise due to pre-analytical variability especially during blood collection and early sample handling. Yi (2008) also reports that in the case of some peptide biomarkers, proteolytic degradation occurs in a matter of seconds.